Pharmaceutical composition comprising the extract of hong-zam as an effective component for prevention or treatment of diabetes and health functional food comprising the same

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of diabetes containing an extract of steamed / lyophilized mature silkworm cultured for 5-instar 7-days as an active ingredient. More specifically, the invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of diabetes containing an extract of steamed / lyophilized mature silkworm cultured for 5-instar 7-days as an active ingredient, obtained by steaming silkworm larvae (*Bombyx mori L.*) grown for 7 days in the 5th instar with water steam and freeze-drying. The extract of steamed / lyophilized mature silkworm cultured for 5-in-1-days as an active ingredient in the pharmaceutical composition for the prevention or treatment of diabetes and health functional food of the present invention is a very useful invention for the pharmaceutical and food industries because it not only has excellent anti-diabetic activity, but also has excellent thermal stability, does not show loss of anti-diabetic activity even under acidic conditions of pH 2 and in plasma, does not show toxicity to the human body, and can be processed into various forms such as extract, powder, pills, and tablets to be prepared in a form that can be taken at any time.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of diabetes containing an extract of *Hongjam* (Steamed / lyophilized Mature Silkworm cultured for 5-instar 7-days) as an active ingredient, and more specifically, to a silkworm grown for 7 days in the 5th instar. Bombyx mori L.) This relates to a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of diabetes containing, as an active ingredient, an extract of red silkworm pupae obtained by steaming the larvae with water vapor and freeze-drying them. Background Technology

[0002] Diabetes, one of the most common diseases among modern people, is a type of metabolic disorder characterized by high blood glucose levels resulting from insufficient insulin secretion or impaired normal function. This hyperglycemia causes various symptoms and signs. Diabetes is classified into Type 1 and Type 2; Type 1 diabetes is caused by the inability to produce any insulin due to genetic factors, while Type 2 diabetes is known to be caused by insulin resistance resulting from impaired insulin function. In particular, environmental factors such as high-calorie, high-fat, and high-protein diets resulting from the Westernization of dietary habits, lack of exercise, and stress are known to play a significant role in Type 2 diabetes. For the treatment of diabetes, insulin injections are essential for Type 1 diabetes, while lifestyle modifications and drug therapy are necessary for Type 2 diabetes; representative medications used include insulin secretagogues (e.g., repaglinide, mitiglinide) and carbohydrate absorption delayers in the small intestine [Glucobay: active ingredient - acarbose; Basen: active ingredient - voglibose].

[0003] Meanwhile, silkworms ( Bombyx moriSilkworms are holometabolite insects that undergo four stages: egg, pupa, and moth. The term "silkworm" typically refers to the "larva of the silkworm moth belonging to the family Bombycidae." Silkworm larvae feed on mulberry leaves throughout their lives, grow rapidly, and can be mass-reared; therefore, they have long been used as sericulture insects, edible insects, and medicinal insects. Recently, as detailed genetic information has become known, they are being used in various genetic and physiological experiments.

[0004] Silkworm larvae and pupae are known to be nutritionally superior, being rich in protein, free amino acids, monounsaturated or polyunsaturated fatty acids, minerals, and fiber; they are already registered as food ingredients in Korea. Furthermore, the efficacy of silkworms is recorded in ancient Korean texts such as the *Donguibogam* and *Bencao Gangmu*, and they have been used in folk medicine for malnutrition, pulmonary tuberculosis, wind pathogens, semen enhancement, and stroke. Recently, with the report of an anti-diabetic active ingredient (deoxynojirimycin), they are gaining attention as a functional food ingredient, and due to advancements in life science technology, they are also being developed as materials for cosmetics and medical applications.

[0005] To date, about 250 varieties of silkworms are known, including Jangchunjam, Bunongjam, Eunbaekjam, Dakwangjam, Daeseongjam, Segwangjam, Samgwangjam, Yangchujam, Yonggangjam, Bunongjam, Baegokjam, Juhwangjam, Yeonnokjam, and Golden Silk. New varieties are continuously being developed not only for the sericulture industry but also for tourism, experiential activities, cosmetic materials, and medicinal purposes.

[0006] Looking at the life cycle of the silkworm, it grows from an egg through five instars. While the duration of the larval period up to the end of the fifth instar varies depending on the variety and environment, it is generally known to be around 20 days. An instar is a term used to distinguish the intervals between molts, and a fifth-instar silkworm refers to a silkworm that has molted four times and emerged. The first-instar silkworm is called the "ant silkworm"; it is very small, initially less than 2 mm in size, but by the fifth instar, it grows to about 8 cm, becoming approximately 8,000 to 10,000 times larger than the ant silkworm. In particular, silkworms consume the most mulberry leaves and grow rapidly during the fifth instar. Typically, starting from the third day of the fifth instar, silk glands develop to build a cocoon, and after the seventh day of the fifth instar, the silkworm stops eating leaves and begins to build a cocoon. At this stage, the silkworm reaches its largest size as a larva, a state known as a mature silkworm.

[0007] Sukjam, which refers to a silkworm just before spinning a cocoon, is a state in which the body is filled with silk protein, appearing clear and transparent. However, the silk protein within the sukjam changes into a very hard structure when dried, making it impossible to chew; attempting to chew it forcefully can damage the teeth, so it exists in an inedible state. Therefore, previously, only silkworms from the 5th instar (3 days) or earlier were used as edible silkworms. However, in order to produce edible sukjam, which is more than twice the size of the 5th instar (3 days) silkworm and has superior nutritional value, the Rural Development Administration has made it possible to consume sukjam after years of research. The method involves rapidly steaming the silkworms to cook them, then freeze-drying them to produce a powder that is easy to digest and absorb. The silkworms produced in this way have been named Hongjam.

[0008] Therefore, the name Hongjam refers to 'steamed, cooked, and dried silkworms' obtained by steaming 5th instar silkworms on their 7th day and then freeze-drying them. It is a name decided through a public contest by the Rural Development Administration in 2017 and carries the meaning of 'silkworms that benefit humans with broad and diverse functionalities.' According to existing reports, Hongjam is known to contain approximately 70% abundant protein, various amino acids, approximately 5% omega-3 fatty acids, various vitamins, as well as various functional components such as flavonoids and polyphenols.

[0009] Studies on fifth-instar silkworms and red silkworm pupae include the antidiabetic activity of edible silkworms (Ryu, KS, et al., 1997. Kor. J. Seric. Sci. 39. 79-85), the antidiabetic activity of silkworm cocoon extracts (Yoon, Ju-hwa et al., 2005. The Korean Society of Food Science and Nutrition 18: 140-145), the development of functional antidiabetic beverages using silkworm extracts (Choi, Jin-ho et al., 2003. Kor. J. Seric. Sci, 45: 96-102), the preparation and quality evaluation of Dacquoise with an optimal ratio of mixed red silkworm pupae and mulberry powder (Kim, Jong-sung, 2019. Master's thesis, Graduate School of Alternative Medicine, Kyonggi University), the general and nutritional components of freeze-dried silkworm powder (Kwon, Hae-yong et al., 2019. Journal of the Korean Society of Sericulture and Entomology 55: 33-39), and recent research on the various health-promoting functionalities of red silkworm pupae. The current status is known (Lee, Hyun-tae, 2019. Korean Journal of Sericulture and Entomology 55: 40-43). In particular, the health functional properties of red silkworm pupae have been confirmed to include preventive effects against Alzheimer's dementia, alcoholic fatty liver, hepatitis and liver inflammation, hyperlipidemia, and hangovers, as well as inhibition of skin darkening caused by ultraviolet rays (Lee, Hyun-tae, 2019. Kor. J. Seric. Sci. 55. 40-43). Furthermore, regarding red silkworm pupae, nutritional component analysis results such as fatty acid, amino acid, and mineral composition have been reported for Baekokjam (white cocoon), Yeonnokjam (light green cocoon), Juhwangjam (red cocoon), and Golden Silk (golden cocoon) (Ji SD et al, 2019. J. Asia-Pacific Entomology 22: 969-974). Regarding blood circulation improvement activity, silkworm powder Bacillus subtilis and Aspergillus kawachi It has been reported that thrombolytic activity is observed when fermented with strains (Cha Jae-young et al., 2011. Journal of Life Sciences 21: 81-88), and platelet aggregation inhibitory peptides have been reported from *Baekgangjam* (White Silkworm Pupa) (Yi Kong et al., 2014. Peptides. 53:70-78). *Baekgangjam* refers to the 4th to 5th instar larvae of silkworm moths larvae infected with the white silkworm pathogen. Beauveria bassianaIt refers to the rigorously dead larvae of silkworm white muscardine caused by natural or artificial infection with *Bals.* Vuill. (Moniliaceae).

[0010] Patents regarding fifth-instar silkworms (5th instar 3 days to 5th instar 7 days) containing red silkworms include Korean Registered Patent No. 10-2286139 [Composition for inducing differentiation of brown adipocytes in canine adipose-derived stem cells using red silkworm extract, method for inducing differentiation, and pet food additive for reducing body fat and preventing obesity containing the same], No. 10-1812499 [Processed food using silkworm powder and method of manufacturing the same], No. 10-0151731 [Hypoglycemic agent containing silkworm powder as an active ingredient and method of manufacturing the same], No. 10-1132337 [Processed flavored oil using silkworm powder and method of manufacturing the same], No. 10-0564202 [Functional health supplement containing silkworm powder extract and method of manufacturing the same], and No. 10-1406109 [Composition for the prevention and treatment of diabetes containing silkworm extract]. Disclosed in Patent No. 10-1246266 [Method for manufacturing fermented silkworm powder and composition for preventing or treating hyperlipidemia and fatty liver containing fermented silkworm powder], Patent No. 10-1359502 [Food composition for preventing and improving osteoporosis using silkworms and health supplement containing the same], Patent No. 10-2270000 [Composition for maturing dendritic cells containing silkworm extract and method for maturing dendritic cells using the same], etc., and Korean Published Patent No. 10-2019-0061544 [Cosmetic composition for antioxidant and whitening containing fermented silkworm powder as an active ingredient] and Published Patent No. 10-2020-0067490 [Method for manufacturing a functional food composition containing silkworm extract] are disclosed.

[0011] However, to date, no reports or patents regarding the potent antidiabetic activity of red silkworm extracts reared for 7 days in the 5th instar have been known. Prior art literature

[0012] Ji SD et al, 2019. J. Asia-Pacific Entomology 22: 969-974Hyuntae Lee, 2019. Kor. J. Seric. Sci. 55. 40-43Ryu, KS, et al. 1997. Kor. J. Seric. Sci. 39. 79-85 The problem to be solved

[0013] The present invention was devised to solve the problems of the prior art as described above, and the problem to be solved by the present invention is to provide a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of diabetes through strong anti-diabetic activity containing steamed / lyophilized mature silkworm cultured for 5-in-star 7-days extract as an active ingredient. means of solving the problem

[0014] To solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing a red silkworm extract as an active ingredient.

[0015] Preferably, the above red silkworm extract is a hot water extract of red silkworm selected from silkworm varieties consisting of white silkworm, golden silk, orange silkworm, and light green silkworm.

[0016] It is preferable that the above red silkworm extract is an ethanol extract of red silkworm, a variety of soft-green silkworm.

[0017] In addition, the present invention provides a health functional food for the prevention or improvement of diabetes containing a red silkworm extract as an active ingredient.

[0018] Preferably, the above red silkworm extract is a hot water extract of red silkworm selected from silkworm varieties consisting of white silkworm, golden silk, orange silkworm, and light green silkworm.

[0019] It is preferable that the above red silkworm extract is an ethanol extract of red silkworm, a variety of soft-green silkworm. Effects of the invention

[0020] The extract of steamed / lyophilized mature silkworm cultured for 5-in-1-days as an active ingredient in the pharmaceutical composition for the prevention or treatment of diabetes and health functional food of the present invention is a very useful invention for the pharmaceutical and food industries because it not only has excellent anti-diabetic activity, but also has excellent thermal stability, does not show loss of anti-diabetic activity even under acidic conditions of pH 2 and in plasma, does not show toxicity to the human body, and can be processed into various forms such as extract, powder, pills, and tablets to be prepared in a form that can be taken at any time. Brief explanation of the drawing

[0021] Figure 1 is a photograph of the four types of red silkworm pupae powder of the present invention. Specific details for implementing the invention

[0022] The present invention will be described in detail below.

[0023] To verify the antidiabetic efficacy of red silkworm pupae, the inventors of the present invention prepared red silkworm pupae powder by steaming and freeze-drying 7-day-old, 5th-instar silkworms of four varieties (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) reared by a specific method. Ethanol and hot water extracts were prepared from this powder, and their antidiabetic activity was evaluated. Consequently, the hot water extracts of red silkworm pupae from Baekokjam, Golden Silk, Juhwangjam, and Yeonnokjam silkworms, and the ethanol extract of red silkworm pupae from Yeonnokjam silkworms were recovered as antidiabetic active components. By confirming that the extracts exhibited excellent thermal and acid stability while showing no hemolytic activity against human red blood cells, the inventors intended to utilize these extracts as pharmaceutical compositions and health functional foods for antidiabetes.

[0024] Specifically, in order to develop pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of diabetes using silkworms, which are known in folk medicine to be effective against various diseases such as diabetes, the vascular and circulatory systems, the digestive system, and the metabolic system, the inventors prepared red silkworms from 7-day-old 5th instar silkworms, the largest in size and most nutritious among the larvae, and prepared various solvent extracts from them. As a result of evaluating their inhibitory activity against α-glucosidase, strong α-glucosidase enzyme inhibitory activity was confirmed in four types of red silkworm hot water extracts and one type of red silkworm ethanol extract. On the other hand, the hot water extract of a conventional 3-day-old 5th instar silkworm powder product showed α-glucosidase enzyme inhibitory activity of 5.2–7.7%, which provided the basis for its use as an antidiabetic agent, but it was weaker than the 14.2–19.4% enzyme inhibition shown by the red silkworm extract of the present invention.

[0025] Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes containing a red silkworm extract as an active ingredient.

[0026] Preferably, the above red silkworm extract is a hot water extract of red silkworm selected from silkworm varieties consisting of white silkworm, golden silk, orange silkworm, and light green silkworm.

[0027] It is preferable that the above red silkworm extract is an ethanol extract of red silkworm, a variety of soft-green silkworm.

[0028] In addition, the present invention provides a health functional food for the prevention or improvement of diabetes containing a red silkworm extract as an active ingredient.

[0029] Preferably, the above red silkworm extract is a hot water extract of red silkworm selected from silkworm varieties consisting of white silkworm, golden silk, orange silkworm, and light green silkworm.

[0030] It is preferable that the above red silkworm extract is an ethanol extract of red silkworm, a variety of soft-green silkworm.

[0031] Below, the method for preparing the red silkworm extract of the present invention and efficacy experiments, etc., will be explained in more detail.

[0032] The present invention comprises the steps of: recovering four types of silkworm larvae (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) reared for 7 days in the 5th instar; preparing an extract by solvent extraction from powder of steamed / lyophilized mature silkworm cultured for 7 days in the 5th instar obtained by steaming the four types of silkworm larvae with water vapor and freeze-drying; evaluating the antidiabetic activity of the extract; and investigating the safety and stability of the steamed silkworm extract.

[0033] The "red silkworm extract" included in the composition of the present invention can be obtained by the steps of: harvesting four types of silkworm larvae reared for 7 days in the 5th instar, drying them, steaming them with steam at 100°C for 15 to 30 minutes, then freeze-drying them to produce a powder, extracting the powder with an organic solvent or hot water, filtering the extract using a filter mesh of 0.06 mm or less, and concentrating the powder under reduced pressure.

[0034] The organic solvent used in the present invention may be water (cold water, hot water), hexene, methylene chloride, acetone, ethanol, anhydrous or hydrated lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, ethanol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc., and hot water or ethanol extraction is preferred.

[0035] The hot water extract or ethanol extract obtained from the above hot water or ethanol extract may be fractionated sequentially or separately with organic solvents of hexene, ethyl acetate, and butanol to additionally obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction, and a water residue.

[0036] In the present invention, hot water extracts and ethanol extracts were prepared for silkworms at 3 days of the 5th instar, which are cultivated and sold in various regions, and for red silkworm larvae of four varieties (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) reared at 7 days of the 5th instar. The α-glucosidase enzyme inhibitory activity was measured by adjusting the extracts to a concentration of 0.5 mg / ml. As a result, strong α-glucosidase enzyme inhibitory activity was confirmed in the hot water extracts of the four varieties of red silkworm larvae and the ethanol extract of Yeonnokjam red silkworm larvae. On the other hand, the hot water and ethanol extracts of silkworms at 3 days of the 5th instar showed relatively weak α-glucosidase enzyme inhibitory activity. Furthermore, unlike the ethanol extract of silkworms at 3 days of the 5th instar which showed strong erythrocyte hemolytic activity, the hot water and ethanol extracts of silkworms at 7 days of the 5th instar did not show erythrocyte hemolytic activity.

[0037] In addition, it was confirmed that the red silkworm extract of the four types of silkworms mentioned above and the red silkworm ethanol extract of the green silkworm have no hemolytic activity against human red blood cells, yet possess excellent thermal and acid stability, allowing them to be consumed or drunk in various forms, and thus can be practically used as a preventive or therapeutic / improving agent for diabetes.

[0038] The red silkworm extract of the present invention can be prepared into a powder through conventional pulverization processes such as vacuum drying, freeze-drying, or spray drying. These are not degraded by various degrading enzymes in plasma and maintain their activity even under heat treatment at 100°C and at a pH of 2 in the human stomach.

[0039] The red silkworm extract of the present invention exhibits strong inhibitory activity against α-glucosidase, and can be used as a material for pharmaceutical compositions and health functional foods related to the prevention, treatment, or improvement of diabetes complications such as type 1 diabetes, type 2 diabetes, diabetic retinopathy, and diabetic nephropathy.

[0040] A pharmaceutical composition containing the active ingredient of the present invention can be formulated into various forms according to conventional methods to suit each intended use, such as oral formulations like powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injectable formulations like sterile injectable solutions, and can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

[0041] These pharmaceutical compositions may additionally include carriers, excipients, or diluents, and examples of suitable carriers, excipients, or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical compositions of the present invention may additionally include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0042] As a preferred embodiment, solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid formulations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above pharmaceutical composition. In addition, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

[0043] As a preferred embodiment, oral liquid formulations may be exemplified as suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients in addition to commonly used simple diluents such as water and liquid paraffin, such as humectants, sweeteners, flavorings, preservatives, etc.

[0044] As a preferred embodiment, formulations for parenteral administration may be exemplified as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

[0045] The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above-mentioned factors into consideration, and this can be easily determined by a person skilled in the art.

[0046] As a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and body weight, and generally, 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0047] The pharmaceutical composition of the present invention may be administered to a subject via various routes. Any mode of administration may be anticipated, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intradural, or intracerebroventricular injection.

[0048] In the present invention, "administration" means providing a specific substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention may be oral or parenteral through any general route capable of reaching the target tissue. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

[0049] In the present invention, "object" includes, but is not specifically limited to, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, and preferably means mammals, more preferably humans.

[0050] In addition, the health functional food of the present invention can be used in various ways, such as in foods and beverages, that are effective in preventing or improving diabetes. Foods containing the active ingredients of the present invention include, for example, various types of food, beverages, chewing gum, tea, vitamin complexes, health supplements, etc., and can be used in the form of powder, granules, tablets, capsules, or beverages.

[0051] The active ingredient of the present invention can generally be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition can be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

[0052] In addition to containing the above compound as an essential component in the indicated proportions, the health functional food of the present invention may contain food-grade acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional components.

[0053] Examples of the above natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, as well as common sugars and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0054] As the above flavoring agents, natural flavoring agents such as stevia, thaumatin, rebaudioside A, or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of the above natural carbohydrates is generally about 1 to 20g, preferably about 5 to 12g, per 100ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various nutritional supplements, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the health functional food of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, vegetable beverages, etc. These ingredients may be used independently or in combination. The proportion of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

[0055] The present invention will be explained in more detail below through examples. The following examples are merely preferred embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[0056] [Example]

[0057] Example 1: Physicochemical characteristics of red silkworm powder from four silkworm larvae (White Silkworm, Light Green Silkworm, Orange Silkworm, and Golden Silk varieties) and commercially available powder from 3-day-old reared 5th-instar silkworms

[0058] Red silkworm powder of commercially available Baekokjam and Golden Silk varieties was purchased from a silkworm rearing farm in Sangju, Gyeongbuk, and for Yeonnokjam and Juhwangjam, which are not currently on sale, red silkworm powder prepared by the aforementioned rearing farm was used. Meanwhile, processed products of 3-day-old 5th instar silkworm powder were purchased from silkworm farms in Suncheon, Yeongcheon, Yecheon, and Sangju.

[0059] First, four types of silkworm larvae (Baekokjam, Yeonnokjam, Juhwangjam, and Golden Silk varieties) reared for 7 days in the 5th instar were steamed at 100°C for 20 minutes, then rapidly cooled and freeze-dried, and then ground to prepare a fine powder of about 50 mesh (Fig. 1). The results of measuring the color difference of the prepared red silkworm powder, as well as their pH and brix, are shown in Table 1, and commercially available silkworm powder reared for 3 days in the 5th instar was used as a control.

[0060]

[0061] [Table 1] pH, Brix, and Color Difference of 4 Types of Red Silkworm Powder and Commercially Available 3-Day 5th Instar Silkworm Powder

[0062]

[0063] As shown in Table 1, the pH of the red silkworm powder from all four silkworm varieties was 6.6; however, regarding brix, which indicates the content of water-soluble substances, the difference was significant in the order of Golden Silk (39.6) > Baekokjam (30.0) > Juhwangjam (26.0) > Yeonnokjam (22.0) (average brix: 29.4±7.54). This indicates that there are differences in the composition depending on the silkworm variety. In addition, as a result of the color difference analysis of the red silkworm powder, the lightness was similar for the Baekokjam, Golden Silk, and Yeonnokjam varieties (51.2–52.8), but the Juhwangjam variety showed a uniquely low lightness of 37.6. The redness was similar for the Baekokjam, Golden Silk, and Yeonnokjam varieties (-2.03–-2.68), but the Juhwangjam variety showed a relatively high redness of 0.10. The yellowness was similar for the Baekokjam and Golden Silk varieties, ranging from 18.12 to 18.26, while for the Juhwangjam and Yeonnokjam varieties, it ranged from 21.22 to 21.32, distinguishing them from the Baekokjam and Golden Silk varieties. Consequently, regarding the overall color difference, Baekokjam and Golden Silk were almost identical, ranging from 43.07 to 43.52, making them difficult to distinguish with the naked eye; Yeonnokjam was characterized by a high yellowness of 45.86, while Juhwangjam was characterized by high redness and yellowness of 58.29. This indicates that there are differences in the pigment components contained depending on the silkworm variety.

[0064] Meanwhile, the pH of commercially available 5th instar 3-day reared silkworm powder showed a slight difference of 6.6–6.9, while the brix showed a significant difference of 38.0–52.8 (average brix: 46.2±6.58). This is understood to be due to the influence of mulberry leaves consumed, which have a brix approximately 1.6 times higher than that of red silkworms reared for 7 days in the 5th instar, as well as the influence of the steaming and drying process. As a result of the color difference analysis of the 5th instar 3-day reared silkworm powder, the lightness was similar at 37.6–44.6, redness at -4.8–-2.1, and yellowness at 16.5–18.7, and the final color difference was similar at 50.4–56.8, showing a distinct difference from the color difference of red silkworm powder.

[0065] Example 2: Preparation of extracts from four types of red silkworms and 3-day-old reared 5th-instar silkworms and analysis of the components of the extracts

[0066] Hot water and ethanol extracts were prepared using the red silkworms of Example 1 and 3-day-old fifth-instar reared silkworms. To prepare the hot water extracts, 20 times the amount of distilled water was added to each silkworm powder sample and extracted at 100°C for 2 hours. To prepare the ethanol extracts, 10 times the amount of ethanol (95%, Deoksan, Korea) was added to the silkworm powder samples, and extraction was repeated twice at room temperature. Subsequently, each extract was collected, filtered, and concentrated under reduced pressure to produce a powder. The extraction yields of the silkworm extracts and the results of the analysis of their useful components are shown in Tables 2 and 3. The total polyphenol content was determined by adding 50 μl of Folin-Ciocalteau and 100 μl of saturated Na2CO3 solution to 400 μl of the extract sample, leaving it at room temperature for 1 hour, and measuring the absorbance at 725 nm. Tannic acid was used as the standard reagent. Total flavonoid content was determined by stirring each sample in ethanol for 18 hours, adding 4 ml of 90% diethylene glycol to 400 μl of the filtered extract, adding 40 μl of 1 N NaOH, reacting at 37°C for 1 hour, and measuring the absorbance at 420 nm. Rutin was used as the standard reagent. Total sugars were quantified using the phenol-sulfuric acid method, with sucrose used as the standard reagent.

[0067] [Table 2] Hot Water Extraction Efficiency and Analysis of Useful Components in Extracts from 4 Types of Red Silkworms and 3-Day-Old 5th Instar Silkworms

[0068]

[0069] As shown in Table 2, the four types of red silkworms showed an extraction efficiency of 21.1–27.0% (average extraction efficiency 24.2±2.48%) in hot water extraction, while the hot water extracts from 5th instar silkworms reared for 3 days showed an extraction efficiency of 22.5–32.1% (average extraction efficiency 27.4±4.58%). These differences in extraction efficiency are understood to be due to silkworm varieties and rearing conditions; overall, the hot water extraction efficiency of 5th instar silkworms reared for 3 days was higher, but no significant difference was observed (p<0.05).

[0070] Meanwhile, analysis of the total polyphenol content of the extracts showed that the hot water extracts of the four types of red silkworms ranged from 18.4 mg / g (Golden Silk) to 39.7 mg / g (Orange Silkworm) (average content 27.1 ± 9.04 mg / g), whereas the hot water extracts of silkworms reared for 3 days at the 5th instar ranged from 29.1 mg / g (Sangju Rearing Farm) to 53.3 mg / g (Yecheon Rearing Farm) (average content 40.7 ± 10.9 mg / g). Regarding total flavonoid content, the four types of red silkworm extracts were similar at 3.4 to 4.4 mg / g, whereas the silkworms reared for 3 days at the 5th instar showed a somewhat significant difference at 4.2 to 16.0 mg / g. Analysis of total sugar content revealed distinctly high levels of over 65 mg / g in the red silkworm extract of the light green silkworm and the extract of silkworms reared in Suncheon for 3 days at the 5th instar.

[0071] [Table 3] Ethanol Extraction Efficiency and Analysis of Useful Components in Extracts from 4 Species of Red Silkworms and 3-Day-Old 5th Instar Silkworms

[0072]

[0073] Meanwhile, as shown in Table 3, the results of the ethanol extraction efficiency and analysis of useful components of the extracts from four types of red silkworms and 3-day-old 5th-instar silkworms showed that the ethanol extraction efficiency was 1 / 2 to 1 / 3 of the hot water extraction efficiency, indicating a low yield, and the total polyphenol content of the ethanol extracts was also found to be 1 / 10 lower than that of the hot water extracts. Specifically, the average total polyphenol content of the ethanol extracts of the four types of red silkworms was 2.75±1.54 mg / g, and the average content of the 3-day-old 5th-instar silkworm extracts was 4.2±3.38 mg / g. The total polyphenol content of the above extracts showed a proportional relationship with antioxidant activity and nitrite scavenging ability.

[0074] Example 3: Evaluation of Antidiabetic Activity of Extracts from 4 Types of Red Silkworm Pupa and 3-Day-Old 5th Instar Silkworms

[0075] The antidiabetic activity of the four types of red silkworm extracts and 3-day-old fifth-instar silkworm extracts prepared in Example 2 was evaluated, and the results are shown in Tables 4 and 5. At this time, the antidiabetic activity of the silkworm extract samples was in-vitro α-glucosidase inhibitory activity was evaluated and expressed. α-glucosidase inhibitory activity was evaluated using pNPG (p-nitrophenol glucoside; Sigma Co., USA). 2.5 μl of silkworm extract sample and 25 μl of α-glucosidase (0.25 U / ml) diluted with 50 mM sodium acetate buffer (pH 5.6) were mixed and subjected to a primary reaction at 37°C for 10 minutes. Subsequently, 25 μl of 1 mM pNPG solution was added and subjected to a secondary reaction at 60°C for 10 minutes. Afterward, the reaction was stopped by adding 25 μl of 1 M NaOH, and the inhibition rate was calculated by measuring the absorbance at 405 nm.

[0076] Inhibition rate (%) = [1 - (Enzyme activity of sample group / Enzyme activity of control group)] x 100

[0077] [Table 4] Antidiabetic activity of hot water extracts of 4 types of red silkworms and 3-day-old 5th instar silkworms

[0078]

[0079] As shown in Table 4, acarbose, which is used clinically as a treatment for diabetes, exhibited potent α-glucosidase inhibitory activity in a concentration-dependent manner. Hot water extracts of the four types of red silkworms showed potent α-glucosidase inhibitory activity of 14.2–16.4%, while extracts from 3-day-old, 5th-instar silkworms also showed enzyme inhibition of 5.2–7.7%. Therefore, it was confirmed that silkworm hot water extracts, particularly the hot water extracts of the four types of red silkworms, can be used for the prevention and treatment of type 2 diabetes. Meanwhile, ethanol extracts of the four types of red silkworms and 3-day-old, 5th-instar silkworms showed weak inhibitory activity, while the ethanol extract of the red silkworms from the soft green silkworms showed α-glucosidase inhibitory activity of 19.4% (Table 5).

[0080] [Table 5] Antidiabetic activity of ethanol extracts from 4 types of red silkworms and 3-day-old 5th instar silkworms

[0081]

[0082] Example 4: Human erythrocyte hemolytic activity of extracts from 4 types of red silkworms and 3-day-old 5th instar silkworms

[0083] To evaluate the potential for acute toxicity of the four types of red silkworm extracts prepared in Example 2 and the four types of extracts from 3-day-old 5th-instar silkworms, human erythrocyte hemolytic activity was evaluated. At this time, hemolytic activity was evaluated according to a previous report (Son Ho-yong, 2014. Korean J. Microbiol. Biotechnol. 42: 285-292). Simply put, 100 μl of human erythrocytes washed three times with PBS were placed in a 96-well microplate, 100 μl of sample solutions of various concentrations were added, and the mixture was reacted at 37°C for 30 minutes. Afterward, the reaction mixture was centrifuged (1,500 rpm) for 10 minutes, 100 μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. DMSO (2%) was used as the solvent control for the samples, and Triton X-100 (1 mg / ml) was used as the experimental control for erythrocyte hemolysis. Hemolytic activity was calculated using the following formula.

[0084]

[0085] [Table 6] Human erythrocyte hemolytic activity of extracts from 4 types of red silkworms and 3-day-old 5th instar silkworms

[0086]

[0087] As shown in Table 6, DMSO and water used as controls showed no hemolytic activity, while Triton X-100 was confirmed to cause 100% hemolysis of red blood cells at a concentration of 1 mg / ml. Additionally, amphotericin B, which is used as an anticancer and antifungal agent, was confirmed to cause more than 50% hemolysis of red blood cells at a concentration of 0.0032 mg / ml. Meanwhile, the four types of prepared red silkworm hot water and ethanol extracts and the hot water extract of 3-day-old 5th-instar silkworms all showed no hemolysis of red blood cells up to a concentration of 1 mg / ml, confirming that they lacked acute toxicity and hemolytic activity. However, the ethanol extracts of 3-day-old 5th-instar silkworms all showed more than 88% hemolysis at concentrations above 0.5 mg / ml, confirming that they contain potential risk factors. These results confirm that the hot water and ethanol extracts of the four types of red silkworms of the present invention can be used for the prevention and treatment of diabetes without exhibiting separate hemolytic activity.

[0088] Example 5: Evaluation of plasma, acid, and thermal stability of hot water extracts of four types of red silkworm pupae and ethanol extracts of light green silkworm pupae

[0089] The silkworms of *Baekokjam*, *Golden Silk*, *Juwangjam*, and *Yeonnokjam*, as well as their *Hongjam*, prepared in Example 2, are used as food, so it is judged that there are no separate safety issues. Accordingly, the plasma stability, heat stability, and acid stability regarding the α-glucosidase inhibitory activity of the hot water extracts of the four types of *Hongjam* and the *Hongjam* ethanol extract of *Yeonnokjam* silkworms were confirmed. The *Hongjam* extracts showed almost no decrease in α-glucosidase inhibitory activity even after heat treatment at 100°C for 1 hour, treatment at pH 2 (0.01M HCl) for 1 hour, and treatment in plasma for 1 hour. Therefore, it can be expected that the extracts contain antidiabetic active substances with acid and heat resistance, and it was confirmed that they have high potential for practical use.

[0090] [Research and Development Project]

[0091] - Supervising Ministry: Ministry of Education

[0092] - Research Management Agency: LINC Project Group

[0093] - Research Project Name: URP and GRP Program

[0094] - Research Project Title: Evaluation of Useful Physiological Activities for the Industrialization of Domestic Red Silkworm Products

[0095] - Contribution rate: 100%

[0096] - Research Period: October 2022 - December 2022 (3 months)

[0097] - Organizing Institution: Industry-Academic Cooperation Foundation, Andong National University

[0098] - Actual research institution: Department of Food and Nutrition, Andong National University

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of diabetes containing, as an active ingredient, a powder prepared by steaming silkworm larvae selected from a group consisting of *Baekokjam*, *Golden Silk*, *Orange Silk*, and *Yeonnokjam*, reared for 7 days in the 5th instar at 100°C for 20 minutes, then rapidly cooling and freeze-drying them, then grinding them to prepare a fine powder of 50 mesh, adding 20 times the amount of distilled water to the fine powder and extracting at 100°C for 2 hours, collecting the extract, filtering it, and concentrating it under reduced pressure, or by steaming silkworm larvae of the *Yeonnokjam* variety, reared for 7 days in the 5th instar at 100°C for 20 minutes, then rapidly cooling and freeze-drying them, then grinding them to prepare a fine powder of 50 mesh, adding 10 times the amount of 95% ethanol to the fine powder, extracting it twice at room temperature, collecting the extracts, filtering them, and concentrating them under reduced pressure. Claim 2 delete Claim 3 delete Claim 4 A health functional food for the prevention or improvement of diabetes containing the active ingredients described in Clause 1.

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